This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding choline phosphate cytidylyltransferase in plants and seeds.
Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) are among the major phospholipids found in plant tissues. The distribution of these lipids among the various organelles of different tissues and among different plants has been comprehensively studied. The pathways by which these lipids are synthesized have also been studied extensively but very few of the plant enzymes involved in these pathways have been purified or their corresponding genes cloned.
Choline phosphate cytidylyltransferase (also called CTP: choline phosphate cytidylyltransferase; E.C. 2.7.7.15) catalyzes the conversion of ethanolamine and choline phosphate to their respective CDP-aminoalcohols. Choline phosphate cytidylyltransferase is thought to regulate the flux through the CDP-choline pathway for PC biosynthesis. In animal and plant cell extracts the choline phosphate cytidylyltransferase enzymatic activity is found in the soluble and in the membrane fractions. It has been proposed that the animal and plant choline phosphate cytidylyltransferases are regulated by the lipid-promoted translocation of the enzyme from the cytosol to the endoplasmic reticulum (ER). In this scenario, the enzyme is inactive while in the cytosole and reversible phosphorylation results in binding to the ER membrane and activation of the enzyme.
cDNAs encoding the rat and yeast choline phosphate cytidylyltransferase proteins have been identified (Kalmar et al. (1990) Proc. Natl. Acad. Sci. USA 87:6029-6033; Tsukagoshi et al. (1987) Eur. J. Biochem. 169:477-486). Pea, rape, and castor bean cDNAs encoding choline phosphate cytidylyltransferases have also been identified (Jones et al. (1998) Plant Mol. Biol. 37:179-185; Nishida et al. (1996) Plant Mol. Biol. 31:205-211; Wang and Moore (1991) Plant Physiol. 96(suppl.):126). Comparison of the amino acid sequences of the rat and yeast choline phosphate cytidylyltransferase show a highly conserved central region surrounded by divergent amino- and carboxy-terminal domains.
The present invention concerns an isolated polynucleotide that encodes a first polypeptide of at least 60 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a second polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:6, 8, 10, 16, and 22. The present furhter concerns an isoalted polynucleotide that encodes a third polypeptide of at least 210 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a fourth polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 14, 18, and 20.
In a second embodiment the first polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21.
In a third embodiment, this invention concerns an isolated polynucleotide encoding a cholinephosphate cytidylyltransferase.
In a fourth embodiment, this invention relates to an isolated complement of the polynucleotide of the present invention, wherein the complement and the polynucleotide consist of the same number of nucleotides and the nucleotide sequence and the complement share 100% complementarity.
In a fifth embodiment, the present invention concerns an isolated polynucleotide that comprises at least 180 nucleotides and remains hybridized to the isolated first polynucleotide of the present invention under a wash condition of 0.1xc3x97SSC, 0.1% SDS, and 65xc2x0 C.
In a sixth embodiment, the invention also relates to a cell comprising an isolated polynucleotide of the present invention. The cell may be a yeast cell, a bacterial cell, or a plant cell. The plant cell may be regenerated into a transgenic plant.
In a seventh embodiment, the invention concerns a method for transforming a cell comprising introducing into a cell the first polynucleotide of the present invention and regenerating a plant from the transformed plant.
In an eighth embodiment, the invention relates to a first isolated polypeptide of at least 60 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a second polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:6, 8, 10, 16, and 22. The invention further relates to a third isolated polypeptide of at least 210 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a fourth polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 14, 18, and 20. The isolated polypeptide may have a sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 14, 16, 18, 20, and 22, and may encode a cholinephosphate cytidylyltransferase.
In a ninth embodiment, the invention concerns a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one regulatory sequence.
In a tenth embodiment, this invention relates to a method of altering the level of a cholinephosphate cytidylyltransferase in a host cell, the method comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in altered levels of the cholinephosphate cytidylyltransferase in the transformed host cell.
A further embodiment.of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a cholinephosphate cytidylyltransferase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a cholinephosphate cytidylyltransferase polypeptide, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of cholinephosphate cytidylyltransferase in the transformed host cell; (c) optionally purifying the cholinephosphate cytidylyltransferase polypeptide expressed by the transformed host cell; (d) treating the cholinephosphate cytidylyltransferase polypeptide with a compound to be tested; and (e) comparing the activity of the cholinephosphate cytidylyltransferase polypeptide that has been treated with a test compound to the activity of an untreated cholinephosphate cytidylyltransferase polypeptide, and selecting compounds with potential for inhibitory activity.